Sample preparation for the detection of infectious agents

ABSTRACT

Methods for improving the quality of sub-optimal patient samples for detection of infectious agents are described. In particular, endocervical fluid samples or vaginal fluid samples are treated with DNase to improve the reliability of detection of infectious agents. Kits for carrying out the methods are also described.

The present invention relates to means for improving disease detection,improved methods for testing for the presence of disease-indicatingmoieties in a test sample and kits for carrying out such methods.

BACKGROUND

It is desirable to perform disease detection tests on patient samples ina manner which provides a reliable disease-specific result and in amanner which does not provide a high level of false positive or falsenegative results. Disease detection may be by detection of a particularanalyte in a sample, for example by detection of a particular antigen orantigenic fragment, antibody or antibody fragment, or nucleic acidsequence. It is preferable if the patient sample requires the minimumamount of preparation and handling prior to testing. It is alsopreferable if the patient sample can be obtained in a completely orrelatively non-invasive manner.

One conventional method for testing for the presence of an analyte in atest solution comprises capturing the analyte on a dipstick anddetecting for the presence of the analyte on the dipstick The dipstickhas a contact end for contacting the test solution and a capture zoneremote from the contact end. The test solution is caused to be drawnfrom the contact end to the capture zone where any analyte present inthe test solution should be captured and an appropriate display providedas a readout. Known types of dipstick use antibody-antigen reactions todetect an analyte. The analyte may be directly indicative of aninfection by an infectious agent or alternatively may be indirectlyindicative of a disease state.

Under controlled test conditions (particularly under laboratory testconditions) it may be possible to carry out detection tests on optimizedsamples. Such samples may be optimized both in terms of the “loading” ofanalyte to be detected (ie the organism load) and also in the qualityand quantity of the test sample in which the analyte is presented fordetection.

In contrast, in many clinical situations, patient samples are collectedin sub-optimal conditions. Furthermore, patient samples may themselvesbe sub-optimal: for example they may have widely varying loadingsbetween samples and/or may be mixed with and contaminated by many othermaterials which may interfere with the test procedure. In addition,these materials may vary widely between individual samples in terms oftheir quantity and/or composition. The individual variation may dependon the physiological condition, health status and/or dietary habit.

As a consequence, some tests which might be proposed to be effective inbuffers may give unreliable results or may fail to work at all whenapplied to real patient samples. An example of this is the detection ofChlamydia trachomatis (CT), the cause of infertility and pelvicinflammatory disease in women. FIG. 1 shows the detection level when theelementary bodies (EB) of CT is spiked in a buffer compared to thatspiked in vaginal fluid. It can be seen that there is a decrease of thetest signal by approximately 100 fold due to inhibitory substancespresent in the vaginal sample.

Manual or automated tests such as Enzyme immunoassays (EIA) rely onsequential steps of incubation with reagents and washing to improve thetest sensitivity and reduce the inhibitory effect of sample on the testresults. However, in the case of dipstick assays, the biological sampleitself serves as a liquid that dissolve the dried reagents impregnatedin the dipstick Because there is no incubation or wash step and becausethe reaction is rapid, the effect of the quality of the sample isparticularly important.

Arko et al (Journal of Clinical Microbiology (1979), 9, 517-519)discloses nuclease enhancement of specific cell agglutination in a slideagglutination test (SAT) for detection of Neisseria gonorrhoeae.However, the test was performed on laboratory cultures (bacterial cellsgrown on agar suspended in PBS buffer containing DNase at 1 mg per ml).There is no disclosure of how to improve testing of patient samples (inparticular of endocervical fluid samples or vaginal fluid samples), norany disclosure that nuclease treatment may enhance tests other than theSAT. Indeed, nuclease treatment did not improve fluorescent staining ofgonococci in a fluorescent antibody test.

Tarkowski et al (Molecular Diagnosis (2001), 6(2), 125-130) describesimproved detection of viral RNA isolated from liquid-based cytologysamples. Total nucleic acid (TNA) was extracted from cell lines grown inthe laboratory (either from fresh cells or from cells fixed inliquid-based cytology media). The extracted TNA was then treated withDNase-I to allow analysis of the RNA in the samples by RT-PCR. However,there is no disclosure of testing of clinical samples, nor of how thesemay be treated to improve the sensitivity, specificity, or reliabilityof the tests.

Hence, there is a desire to provide improved disease detection testswhich are capable of performing to an acceptable level of sensitivity,specificity, reliability and ease of use on patient samples irrespectiveof the “quality” of the sample.

BRIEF DESCRIPTION OF THE INVENTION

According to the present invention there is provided a means forimproving the “quality” of sub-optimal patient samples for detection ofinfectious agents. The invention provides several steps which can betaken alone or in combination which result in improved test performanceby sub-optimal patient samples.

In a preferred aspect, the invention provides a method for treatment ofa human patient sample for carrying out a diagnostic method on thesample for detection of an infectious agent, wherein the sample is anendocervical fluid sample or a vaginal fluid sample, which includes thestep of carrying out the diagnostic method in the presence of DNase.Preferably the endocervical fluid sample or the vaginal fluid sample istreated with DNase.

It will be appreciated that the invention does not require a priorknowledge of the quality of the patient sample: samples which are ofpoor quality will be improved and samples which are already of highquality might be unaffected or marginally improved. The overall effectis that a highly reliable test result can be obtained on patient samplesirrespective of the original quality without the need for qualitytesting.

The invention will now be described in detail with regard to testing forthe sexually transmitted disease caused by Chlamydia trachomatis by theanalysis of self-collected vaginal swabs as the sample type. However,the invention is not to be construed as being limited to the preferredexamples, and is only defined in scope by the claims.

The Importance of Chlamydia as a Disease

CT is the most common sexually transmitted pathogen in westerncountries. CT serotypes D-K have a worldwide distribution and are thecause of genital infection and associated ocular and respiratoryinfections. Chlamydia infection may lead to serious complications andsequelae: for example Pelvic Inflammatory Disease (PID), ectopicpregnancy, infertility in women, and perinatal and congenital infectionsin babies born from infected mothers.

CT infections often remain undiagnosed because many infected women havemild, non-specific symptoms or are asymptomatic. Screening for CTinfection is a critical component of control efforts. Diagnosis of CTinfection in women using EIA or rapid tests currently requires a pelvicexamination. Only amplified nucleic acid tests such as Ligase ChainReaction (LCR) or Polymerase Chain Reaction (PCR) are capable of testingurine samples. Testing with LCR or PCR is complex, requires expensiveinstruments and the test result is not usually available until a weekafter the taking of the sample. The use of non-invasive samples (i.e.urine or self-collected vaginal swabs) with rapid tests could allowtesting of more women including those who are either deterred by apelvic examination or do not have access to one (i.e. due to lack ofaccess to a trained medic or physician).

LCR testing of paired urine and vaginal swab samples from womenattending a genitourinary clinic have shown that there are lower countsof CT organism in urine compared to vaginal fluids. Therefore, the useof self-collected vaginal sample has the advantage of being anon-invasive sample type that is simple to collect. Due to its higherorganism load the use of this sample would also improve the testsensitivity compared to urine.

Current protocols for urine testing require a dilution step with waterbefore centrifugation to reduce the inhibitory effect of urine to thetest. In addition, the use of urine as a sample requires a concentrationstep, which not only adds to the cost but to the processing time. Oneexample of this is the Clearview Chlamydia ME (Unipath LTD, Bedford,England) test where 10ml of urine specimen is diluted with an equalvolume of distilled or deionised water before centrifugation at 3000 gfor 15 minutes.

Although no current test has a product claim for vaginal swab sample, wehave found that vaginal swab samples are inhibitory to currentantibody-based or amplified DNA Chlamydia tests. These antibody-basedtests include Clearview Chlamydia MF, QuickVue Chlamydia (Quidel Corp,CA, USA), and Chlamydia OIA (Biostar, CO, USA).

The Clearview Chlamydia MF test is a lateral flow immunoassay intendedfor detection of Chlamydia trachomatis antigen in either femaleendocervical swab specimens or male urine specimens. The male urinespecimens require dilution and centrifugation step as mentioned abovewhile the swab specimens do not. Extraction involves addition ofextraction reagent, heating at 80° C. for 10 minutes, and allowing thesample to cool for at least 5 minutes. Test procedure involves applying5 drops of the sample extract to the sample window of the test unit(immunochromatographic test strip) and reading the result after 15minutes.

The QuickVue Chlamydia test is a lateral flow imununoassay intended fordetection of Chlamydia antigen from endocervical swab and cytology brushspecimens. To perform the test, an endocervical clinical specimen isobtained and placed into a tube containing the extraction solution;after 2 minutes, neutralization solution is added to the tube. Afterextraction and neutralization, 3 drops of extracted sample is added tothe sample well of the test cassette and the results read after 10minutes.

The Chlamydia OIA is an optical immunoassay intended for detection ofChlamydia antigen from female endocervical swabs and neonatalconjunctival specimens. The test involves the extraction of ChlamydiaLipopolysaccharide (LPS) from the specimen using two specimen extractionreagents and a neuralization reagent, and the subsequent use of OpticalImmunoassay technology for the qualitative detection of the antigen. Thedetection or testing stage involves five steps: application of thesample extract to the test device (5-min incubation), addition of theconjugate (5-min incubation), washing, addition of the substrate (5-minincubation), and a final wash. Test results are evaluated under a brightlight source with light reflected off the test surface.

We have developed a rapid immunochromatographic strip test for thedetection of Chlamydia LPS antigen using self-collected vaginal swabs.To perform the test, the swab specimen is placed into a tube containingthe extraction solution. The sample extract is neutralized, and analiquot is applied to an immunochromatographic test strip (i.e. a“dipstick”). The test results are read after 15-25 minutes.

Sample Preparation

An inhibitory effect of vaginal fluid on the assay sensitivity wasobserved when known amounts of EB's were spiked into vaginal swabs (FIG.1). The signals generated in the present of vaginal fluid showed areduction of approximately 100 fold compared to buffer. There are atleast two aspects to the inhibitory phenomenon observed with vaginalswab specimens: direct inhibition of antibody-antigen interaction andindirect inhibition of the test by preventing proper mixing of reagentsand the reduction or inhibition of liquid flow.

The vaginal sample contains components that directly inhibit theinteraction between antibodies and their target antigen (i.e. LPS andanti-LPS antibodies). This inhibition may be through physically blockingthe antibody and antigen from coming together, sequestration of the LPStarget, or modification of charges on the antibody molecule adverselyaffecting its affinity. The inhibitory effect varies widely betweenindividuals and within the same individual during different periods ofthe menstrual cycle.

Inhibition of proper mixing and liquid flow is related to the inherentviscosity of vaginal fluid. Two of the main contributors to thisviscosity are mucin levels and amounts of DNA in the sample. The highmucin level in some samples contributes to the viscosity and largeamounts of DNA tend to form a gel-like matrix thus physically clog themembrane and prevent or reduce fluid flow.

Vaginal fluids are highly variable from individual to individual andduring different periods within a woman's monthly menstrual cycle. Thepresence of other things like sperm, excessive bacterial growth, yeastinfection, vaginal douches, and lubricants may also contribute to thisvariability.

GENERAL EXAMPLE: Vaginal Swab Samples

Sample collection

-   1. Use either a polyurethane or polyester (e.g. Dacron) swab (on a    polystyrene or polypropylene plastic shaft) to collect the vaginal    sample. Tampons and sanitary napkins may also be used for sample    collection.-   2. The swab should be inserted preferably 6 cm (3 cm to 9 cm) deep    from the opening of the vagina and rotated several times for at    least 10 seconds before removal.-   3. The sample can either be stored dry or in sample collection    buffer at 2-8° C. for 2-4 days before testing.    Sample preparation    1. Use of DNase to degrade nucleic acids present

Some vaginal swab samples contain large amounts of DNA which forms agel-like matrix that tend to retain fluid, clog the nitrocellulosemembrane and inhibit the migration of reagents, and results in a totalfailure of the test. Digestion of the DNA with DNase prevents the abovefrom happening. DNase is effective when added at more than 0.5 jig/ml or1.5 units of activity per ml, for example 0.5-100 μg/ml or 1.5-300 unitsof activity per ml. The required amount of DNase ultimately depends onthe amount of DNA in the sample and the length of time given for theenzyme to act.

FIG. 4 shows the beneficial effects of DNase treatment on the dipstickassay. In the vaginal fluid of an individual infected with Chlamydia,treatment with DNase prevented clogging at the bottom of the strip andallowed a stronger positive signal to develop at the test line. In theChlamydia negative vaginal swab sample B, clogging at the bottom of thestrip was prevented by DNase treatment, enabling the procedural controlsignal to develop.

Neuraminidase and lysozyme were also tested along with DNase for theireffect on the inhibitory property of vaginal fluid. Unlike DNasehowever, neither enzyme consistently affected the inhibitory phenomenonof vaginal fluid. Neuraminidase was tested from 3 to 200 units ofactivity per ml of vaginal sample extract while lysozyme was tested at830 to 100,000 units per ml of vaginal sample extract.

2. Use of n-dodecyl maltoside as a surfactant

The non-ionic alkyl glucosides, particularly n-dodecyl maltoside, arethe most effective surfactants for extracting CT LPS from vaginalsamples and making it available for antibody detection. N-dodecylmaltoside works best at concentrations of 0.01% to 0.04% w/v, preferably0.015% to 0.03% w/v.

Table 1 shows that when vaginal swabs were taken from differentindividuals (A to G), spiked with 20,000 Chlamydia EB per swab, theaddition of n-dodecyl maltoside to the extraction buffer gave the bestsignal results.

3. Use of PVA or PVP

Polyvinyl alcohol (PVA) is an effective blocking agent when usingvaginal swab samples. PVA is thought to coat the nitrocellulose membranefibres, effectively blocking it from binding other reagents. This allowsmore reagent to be available for reaction at the capture line. It alsoresults in a cleaner background for the test. In addition, it enhancesthe sensitivity of the dipstick test either by acting as an LPS carrieror by enhancing the formation of LPS micelles. PVA works best atconcentrations of 0.01% to 0.5% w/v. Polyvinyl pyrrolidone (PVP) alsoenhances the sensitivity of the test through a mechanism similar to thatof PVA and works best at 0.2% to 2% w/v.

Table 2 shows that when vaginal swabs were taken from differentindividuals (A to G), spiked with 20,000 Chlamydia EB per swab, andtested with or without PVA in the extraction solution, the signals werestronger in those extracted in the presence of PVA.

4. Use of H₂O₂ to oxidize inhibitory substances

The addition of H₂O₂ is able to neutralize some of the inhibitoryeffects of vaginal samples. This effect may be associated with H₂O₂ asan oxidizing agent and therefore neutralizing some of the inhibitorysubstance(s) by oxidation. The optimum concentration of H₂O₂ is between0.5% to 3% w/v. It becomes less effective below this range and begins toadversely affect the test above this range.

FIG. 2 shows the effect of H₂O₂ on the signal of a CT dipstick test indifferent individuals. Vaginal swabs were collected from four differentindividuals (A to D) and spiked with 90,000 Chlamidia EB per swab Theextraction step was carried out with or without the addition of 1% H₂O₂. It can be seen that in samples not treated with H₂O₂ , no positivesignal was visible whereas in those treated with H₂O₂ all yielded strongpositive signals.

As demonstrated by FIG. 3, the inhibitory substance(s) exist mainly inthe soluble fraction of the processed samples. Vaginal swabs taken fromindividuals A-D were spiked with 80,000 EB's per swab. After extractionof LPS, half of the samples were further clarified by centrifugation toremove particular matters. Both centrifuged and non-centrifuged sampleswere tested for the presence of LPS with or without H₂O₂ treatment. Itcan be seen that a marked improvement of signal strength was observed inall samples with H₂O₂ treatment.

General example of a sample preparation procedure:

A self-collected vaginal swab specimen is first obtained and thentreated in the following manner. (Note that if a tampon or sanitarynapkin is used to collect the specimen, the amounts of reagents usedwill have to be adjusted accordingly.)

-   1. Add 400 μl reagent A to the swab to disrupt EB and extract LPS.    Reagent A contains 100 to 300 mM NaOH. The swab should be allowed to    incubate in reagent A for no longer than 5 minutes.-   2. Add 300 μl reagent B to decrease the pH, provide a protein and    polymer blocker, and form LPS micelles. Reagent B is a 0.5M Tris    buffer, pH8.5 containing 100 mM NaCl, 130-400 mM HCl and 1%-4%    protein such as bovine serum albumin, 0.03%-1.3% polyvinyl alcohol    and 0.03%-0.1% n-dodecylmaltoside. The swab should not be allowed to    incubate for more than 5 minutes.-   3. Add 100 μl reagent C to oxidize inhibitory substances. Reagent C    is 6% H₂O₂. The sample should not be allowed to incubate for more    than 2 minutes.-   4. Apply the sample to the test strip in the presence of more than    0.5 μg/ml or 1.5 units of activity per ml, for example 0.5-100 μg/ml    or 1.5-300 units of activity per ml DNase.    References

Smith K, Harrington K, Wingood G, Oh MK, Hook EW, DiClemente RJ. 2001.Self-obtained vaginal swabs for diagnosis of treatable sexuallytransmitted diseases in adolescent girls. Arch Pediatr Adolesc Med155:676-679. TABLE 1 Individuals Surfactants A B C D E F G n-Dodecylmaltoside 1.5 2 2 1.5 2 2 2 n-octyl glycopyranoside 1 1.5 0.5 1 1.5 2 2Tween 20 2 0.5 1 0.5 1 1.5 2 Chapso 0.5 0.5 0.5 0.5 1 1 1 Cholate 0.50.5 1 0.5 0.5 1 1The signals are on a scale of 1 to 5 with 5 being the strongest signal.

TABLE 2 Individuals Treatment A B C D E F G Without PVA 1 1.5 1 1.5 10.5 0.5 With PVA 2 2.5 2 2.5 2 2.5 1.5The signals are on a scale of 1 to 5 with 5 being the strongest signal.

1. A method for treatment of a human patient sample for carrying out adiagnostic method on the sample for detection of an infectious agent,wherein the sample is an endocervical fluid sample or a vaginal fluidsample, which includes the step of carrying out the diagnostic method inthe presence of DNase.
 2. A method according to claim 1 wherein theDNase is present in an amount of more than 0.5 μg/ml, preferably 0.5 to100 μg/ml.
 3. A method according to claim 1 wherein the DNase is presentin an amount of more than 1.5 units of activity per ml, preferably 1.5to 300 units activity per ml.
 4. A method according to any of claims 1to 3 which additionally includes a method for preparation of a humanpatient sample prior to carrying out a diagnostic method on the samplefor detection of an infectious agent, which preparative method includesthe step of treating the sample with an oxidizing agent.
 5. A methodaccording to claim 4 wherein the oxidizing agent is hydrogen peroxide(H₂O₂).
 6. A method according to claim 5 using a working concentrationof hydrogen peroxide of 0.5% to 3% w/v.
 7. A method according to any ofclaims 1 to 3 or 4 to 6 which additionally includes the step of treatingthe sample with a non-ionic alkyl glucoside surfactant.
 8. A methodaccording to claim 7 wherein the surfactant is n-dodecyl maltoside.
 9. Amethod according to claim 8 wherein the n-dodecyl maltoside is presentat a working concentration of 0.01% to 0.04% w/v, preferably 0.015% to0.03%.
 10. A method according to any of claims 1 to 3 or 4 to 6 or 7 to9 which additionally includes the step of treating the sample either orboth of PVA and PVP.
 11. A method according to claim 10 wherein whereinthe sample is treated with PVA, preferably having an average molecularweight between 20 and 25 kDa and at a working concentration of between0.01 and 0.5% w/v.
 12. A method according to claim 10 wherein the sampleis treated with PVP at a working concentration between 0.2% and 2% w/v.13. A method according to any of claims 1 to 3, which additionallycomprises a method step according to any of claims 10 to 12 and a methodstep according to any of claims 7 to 9 and a method step according toany of claims 4 to
 6. 14. A method according to any of claims 1 to 13wherein the human patient sample is obtained as a self-collected vaginalswab sample.
 15. A method according to any of claims 1 to 13 wherein themethod is for detection of Chlamydia trachomatis.
 16. A method accordingto any of claims 1 to 13 wherein the patient sample is a self-collectedvaginal swab sample and the method is for detection of Chlamydiatrachomatis.
 17. A method according to any preceding claim wherein themethod is a dipstick test method.
 18. A kit comprising: a dipstick testapparatus for carrying out a specific infectious agent detection test;reagents required for said apparatus in order to carry out said specificdetection tests; a DNase reagent for carrying out the method of any ofclaims 1 to
 3. 19. A kit according to claim 18 additionally comprising:an oxidizing agent reagent for carrying out the method of any of claims4 to
 6. 20. A kit according to claim 18 additionally comprising: anon-ionic alkyl glucoside reagent for carrying out the method of any ofclaims 7 to
 9. 21. A kit according to claim 18 additionally comprising:a reagent which is PVA and/or PVP for carrying out the method of any ofclaims 10 to
 12. 22. A kit according to claim 18 additionallycomprising: a non-ionic alkyl glucoside surfactant reagent as defined inany of claims 4 to 6 and a PVA and/or PVP reagent as defined in any ofclaims 7 to 9 for carrying out the method of any of claims 1 to 15.